WO2002074898A2

WO2002074898A2 - Gradient block temperature control device

Info

Publication number: WO2002074898A2
Application number: PCT/IB2002/001828
Authority: WO
Inventors: Michael Hodges
Original assignee: Techne (Cambridge) Ltd
Priority date: 2001-03-16
Filing date: 2002-03-18
Publication date: 2002-09-26
Also published as: WO2002074898A3

Abstract

This invention relates to a temperature control apparatus for generating and maintaining a differential temperature gradient across a block that may be used, for example, to conduct biological reactions. According to one embodiment of the present invention, a gradient block includes a top plate and a data storage device. The data storage device is adapted to be connected to a control unit and stores data related to the calibration of the gradient block. According to another embodiment of the present invention, an apparatus for generating a temperature gradient across a gradient block includes a gradient block and a control unit for controlling the temperature of the thermoelectric devices. The control unit includes a top plate, and at least four pairs of thermoelectric devices in thermal contact with the top plate, the pairs of thermoelectric devices being capable of independently transferring heat with the top plate and dividing the top plate into at least four heat transfer zones. The control unit contains a microprocessor capable of storing data.

Description

GRADIENT BLOCK TEMPERATURE CONTROL DEVICE

BACKGROUND OF THE INVENTION

Field of the invention

This invention relates to a temperature control apparatus for generating and maintaining a differential temperature gradient across a block that may be used, for example, to conduct biological reactions.

Related Art

Thermal cyclers are instruments which are used to conduct biological reactions, such as the Polymerase Chain Reaction (PCR). PCR involves multiple heating and cooling steps in a single complete thermal cycle to sequentially denature DNA strands contained in a sample, to anneal primers to the denatured DNA, and to extend the annealed primers. Temperatures for performing these process steps depend on the composition of the individual samples, and are determined experimentally by the researcher. The PCR reaction, in particular, is known to be sensitive to the sample temperature and the time the sample is held at a particular temperature. The reaction can fail completely if the temperature is too low or too high, or if the time the sampleis maintained at a given temperature is too long or too short.

Once a general temperature range is established, optimal temperatures for the reaction can be determined. Optimization of process conditions is critical for reaction efficiency and specificity. The optimal temperature depends on many factors, including the length and type of DNA involved in the PCR process, the length and composition of the primer, and the type of enzyme used in the process. The optimal temperature for each of the process steps is essentially independent of the optimal temperature for the other steps. However, the annealing step is considered to be the most critical for optimization of the PCR reaction.

The use of a gradient block permits the operator of the thermal cycler to conduct multiple PCR experiments by permitting the operator to vary the temperature across the block. Prior to the advent of the gradient block, thermal cyclers had used uniform temperature blocks which maintained the reaction temperature for the individual process steps at uniform levels for all sample tubes. This made the determination of the optimum temperature for the reaction an exceedingly complex and time consuming process.

Many of these problems involving optimization are solved by thermal cyclers which incorporate a temperature gradient block. These thermal cyclers permit the simultaneous reaction of multiple biological samples at temperatures within a preselected range to identify the optimum temperature for a given PCR reaction.

U.S. Patent No. 5,525,300 and U.S. Patent No. 5,779,981, the respective disclosures of which are incorporated herein by reference in their entireties, describe an apparatus and method for generating a temperature gradient across a gradient block. The gradient block apparatus contains a heater at one end of the block, and a cooling device at the opposite end of the block. The gradient is established by the temperature differential maintained across the block by the heater and cooler which contact the ends of the block. The block has holes drilled vertically in the top surface for holding sample tubes. Typically, the block contains 96 such holes in an 8x12 rectangular configuration. Sample tubes containing liquid samples of DNA are placed in the holes, and the PCR protocol is programmed into the computer control system which regulates the temperature of the heater and cooler, and creates a gradient across the block.

PCT published patent application WO 98/20975 discloses a temperature regulating block for controlling the temperature of laboratory instruments. The top of the block contains holes for sample containers for containing liquids of biological fluids. The bottom of the block contains at least two temperature-regulating devices which are in thermal contact with the block to produce different temperatures in different sections of the block.

These various devices, while relatively effective compared to thermal cyclers which are not equipped with gradient temperature blocks, still suffer from certain drawbacks. For instance, certain devices do not have accurate mechanisms for insuring that the temperature across the block is strictly linear, which is generally regarded as the ideal condition. This can result in the establishment of a non- linear gradient along the length of the block, which can typically take the form of an "S" shaped curve when the temperature is plotted against distance along the block. Such a non-linear gradient is undesirable since the temperature cannot be accurately fixed by the temperature controller. Attempts to compensate for this temperature variation typically involve auxiliary heating and cooling devices, or structural modifications to the block. For instance, the block described in U.S. Patent No. 5,525,300 utilizes a series of holes drilled horizontally through the block below the upper surface of the block. The intent of these holes is to limit thermal conductivity so that each row of holes can be maintained at a uniform temperature, and the temperature gradient can approach uniform linearity.

Thermal cyclers, such as those described in U.S. Patent No. 5,525,300, are incapable of generating a temperature profile other than a substantially linear gradient. While this is perfectly acceptable for the vast majority of practical applications, additional flexibility is useful for applications which may require or benefit from a temperature profile having a lower (or higher) temperature in the interior portion of the block than at either of the ends. Such a profile would not be possible using the device described in U.S. Patent No. 5,525,300.

SUMMARY OF THE INVENTION

According to one embodiment of the present invention, a gradient block is provided. The gradient block includes a top plate and a data storage device. The data storage device is adapted to be connected to a control unit and stores data related to the calibration of the gradient block. According to another embodiment of the present invention, an apparatus for generating a temperature gradient across a gradient block is provided. The apparatus includes a gradient block and a control unit for controlling the temperature of the thermoelectric devices. The control unit includes a top plate, and at least four pairs of thermoelectric devices in thermal contact with the top plate, the pairs of thermoelectric devices being capable of independently transferring heat with the top plate and dividing the top plate into at least four heat transfer zones. The control unit contains a microprocessor capable of storing data.

According to another embodiment of the present invention, a method for replacing a gradient block in a thermal cycler is provided. The thermal cycler for use in this method includes a computer and a first gradient block. The method includes removing a first gradient block from the thermal cycler, connecting a second gradient block comprising a second data storage device containing thermal calibration data, and modifying a thermal control algorithm of the computer to reflect the thermal characteristics of the second gradient block.

According to another embodiment of the present invention, a method of using a thermal cycler is provided. The method includes installing a gradient block onto the thermal cycler, providing data stored on a data storage device connected to the gradient block to a control unit connected to the thermal cycler, and controlling at least one heat transfer device connected to the gradient block based on the data provided.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is schematic view of one aspect of the present invention;

FIG. 2 is a perspective view of one embodiment of the present invention; and FIG. 3 is a plot of the temperature profile of the gradient block of the present invention and the temperature profile of a gradient block of a commercial thermal cycler.

DETAILED DESCRIPTION

In one embodiment, the apparatus of the present invention features a gradient block, including a top plate and at least 4 pairs of thermoelectric heating and cooling devices in thermal contact with the top plate for heating and cooling the top plate. In this embodiment, the apparatus further includes an electronic control unit including a microprocessor for storing temperature and reaction cycle data. As used herein, "top plate" refers to a piece of material adapted to transfer heat to or from heating or cooling devices. Also as used herein, "gradient block" refers to the top plate and related structures such as heating or cooling devices, sensors, heat sinks, and the like. Typically, a gradient block will have a varying temperature profile across it in a lengthwise direction. However, it should be understood that this term, as used herein, is also applied to blocks having a constant temperature.

Where the thermoelectric devices are used in the gradient block, they may be positioned so as to provide a desired temperature profile, such as a substantially linear temperature profile. For example, the thermoelectric devices may be in a paired relationship, with each member of the pair being positioned on opposite sides along the width of the block. The paired thermoelectric devices may be arranged in a column along the length of the block. Each member of the pair may be electrically connected in series, operating as a unit. However, the individual pairs may also be designed to operate independently, and may be able to be programmed separately. This arrangement of heating and cooling devices permits a wide temperature range of up to 30°C or more across the block when a linear gradient is desired. Further, four independently controllable zones enable an apparatus according to the present invention, such as a thermal cycler, to span at least the ambient temperature range of 20°C to 40°C.

In embodiments having heating and cooling devices arranged across the length of the block, a more linear gradient is possible than with conventional thermal cycling devices. Typical deviations from linearity of +0.5°C or less can be achievable in the gradient block of the present invention, compared with deviations of +1.5°C or more in conventional gradient blocks.

Placement of heating and/or cooling blocks in a lengthwise pattern along a top plate may divide a gradient block into heat transfer zones. These heat transfer zones may allow, for example, the interior of the gradient block to be maintained at a lower (or higher) temperature than the temperature at either ends of the gradient block. Thus, in this embodiment, the thermal cycler of the present invention is capable of producing non-linear temperature profiles in the gradient block if desired. Alternatively, this embodiment of the thermal cycler of the present invention may be operated in a constant temperature mode, if desired, meaning that it can maintain a gradient block as a constant temperature block.

FIG. 1 illustrates an example placement of thermoelectric devices in a pian view of a gradient block 10. The arrows in the diagram indicate pairs of physically opposed thermoelectric devices. A first thermoelectric device 20 is shown in a paired, opposed relationship to a second thermoelectric device 30. The drawing illustrates four such pairs of devices. As shown, the gradient block is effectively divided into four separate zones of pairs of thermoelectric devices. Each zone may be electrically independent of the other zones, and may be operated separately if desired.

It should also be appreciated that the use of thermoelectric devices is a preferred embodiment of the present invention, but that other devices could be used. Any heat transfer device that is able to heat and/or cool quickly enough, provide adequate temperature resolution, and provide adequate temperature accuracy for a particular use may be useful instead of a thermoelectric device in the present invention. Where a thermoelectric device is used in the present invention, preferred thermoelectric devices include Peltier devices. Peltier devices are electrical or electronic devices which transfer heat from one circuit junction having conductors made of different materials, to another junction in the same circuit in response to a direct current. Depending on the direction of the current, the junction from which heat is removed is the cold source, and the junction which receives heat is the hot source. These devices can be embedded in the gradient block and may act as heat pumps which raise or lower the temperature of the top plate depending on the direction of the applied current.

The top plate of the present invention may be any thermally conductive piece of material. In one embodiment, the top plate may be square or rectangular. The top plate is preferably fabricated from a heat conducting metal such as aluminum, brass or silver, and may have holes in the top for accommodating tubes containing reagent samples. Preferably, the top plate has 96 such holes in an 8x12 configuration, although blocks having 384 holes in a 16x24 configuration, or even more holes, in some cases arranged irregularly, may also be used. Alternatively, the block may have fewer holes, for instance 40 holes adapted for holding 0.5ml tubes, or no holes, i.e. a flat plate.

The gradient block may include one or more sensors. Sensors may be positioned on, or in, the gradient block, such as within the top plate. For example, the gradient block may include a plurality, such as four, temperature sensors for recording the temperature at various locations along the top plate. The use of a plurality of temperature sensors may permit a more accurate temperature profile to be used. In addition to a gradient block, a thermal cycler according to the present invention preferably contains an electronic control unit. The electronic control unit may include a microprocessor, which may be capable of storing data, such as temperature data collected from any temperature sensors, and data regarding temperature protocols for particular thermal cycles, such as PCR reactions. The microprocessor may also be used to program temperature profiles and reaction times of samples based on input data from the operator. An electronic control unit including a microprocessor is also referred to as a computer herein. In a preferred embodiment, input data is supplied by the operator through a touchscreen which communicates directly with the microprocessor. The microprocessor may send commands to thermoelectric heaters and/or coolers in the gradient block. The touchscreen may also act as a graphical display for displaying the temperature profile across the block in real-time output. This arrangement may provide a convenient and user friendly method for visualizing the progress of an experiment in real time.

In a typical PCR reaction, the operator places tubes containing a nucleic acid reaction mixture into sample tube holes in the top of the block. A typical 96 hole configuration, for instance, involves the arrangement of 8 rows and 12 columns of tubes. Each column of tubes is maintained at a constant temperature, with the temperature of each row varying along the length of the block. Typically, the temperature of a column varies between one and two degrees from the next adjacent column in a linear gradient. A graph showing the typical temperature profile of the gradient block of this invention is shown in FIG. 3. This is contrasted with the temperature profile for a conventional gradient block as shown in FIG. 3. The gradient of the present invention is seen to be significantly more linear than the conventional apparatus.

In typical operation of an example embodiment of the present invention, the operator programs a non-optimized temperature range for a selected PCR reaction into the thermal cycler microprocessor software. The end points of the temperature ranges for the annealing step of this reaction, and the corresponding reaction time for this step, are identified and programmed into the microprocessor. The end points of the temperature range are used to set the temperature ranges at each end of the gradient block.

The samples are placed in the gradient block, the temperature range is set, and the temperature gradient is held for the time period indicated in the PCR protocol. The samples are then analyzed for reaction yield and efficiency, and the optimal temperature and reaction time for each step of the PCR reaction is determined based on the condition of the samples.

The optimization data is programmed into the microprocessor, which can then be programmed for the desired number of PCR cycles required for the particular PCR protocol. Each cycle is essentially a repeat of the previous cycle, and does not require additional monitoring by the operator. A typical protocol requires on the order of 30 PCR cycles, and usually takes 1.5 to 2.0 hours or more to complete.

Operation of the thermal cycler following identification of the optimal temperatures typically occurs in a fixed temperature mode. The temperatures established during the optimization procedure are programmed into the microprocessor as fixed temperature settings for each PCR process step. If desired, a larger number of samples than the conventional 96 sample arrangement may be used by using a gradient block with a larger number of sample tube holes. The thermal cycler is then run in automated mode without the need for additional operator input. Information concerning the progress of the PCR cycling reaction is displayed on the graphical interface output screen. In a typical cycle, the temperature of the gradient block is raised to an elevated temperature of, for instance, 90°C, and held at that temperature for sufficient time to denature the DNA in the sample. The temperature of the block is then lowered to a temperature of about 50°C and held at that temperature for sufficient time to anneal the denatured, single-stranded DNA to primers which have been added to the sample. Following annealing of the primers, the temperature of the block is then raised to a temperature of about 70°C and held at that temperature for sufficient time to extend the annealed primers. Primer extension is initiated in the presence of a polymerase enzyme, such as a Taq polymerase enzyme, which is included in the sample. The enzyme must be capable of withstanding the elevated temperatures which occur during the PCR cycle.

The cycle is repeated by the thermal cycler in an automated mode for the number of cycles programmed into the microprocessor. At the end of the final cycle, the sample tubes are removed and the amplified DNA is collected. The DNA in the sample increases geometrically after each cycle, and the final concentration of DNA in the sample tubes is usually on the order of a billion times greater than the initial DNA concentration.

In one embodiment of the present invention, substitution of one gradient block for another is facilitated by a data storage device associated with the block and able to store a record of at least some of the thermal characteristics of the block, easing calibration. For example, in a preferred embodiment, non- volatile memory, such as a flash memory chip, may be included with the block. In one example embodiment, data stored in the microprocessor regarding the expected temperature error rate can be compared to similar data contained in the data storage device for the new block, and thereby used to simplify the calibration of the new block. It should be understood that the use of a data storage device positioned in a gradient block to store calibration information about the gradient block is applicable to any gradient block, and not just the embodiments disclosed herein. It also bears repeating that the term "gradient block" as used herein includes constant temperature blocks, including blocks having only a single heating or cooling device. An apparatus according to the present invention, such as a thermal cycler, may be housed in a compact desk top plastic housing as shown in FIG. 2. FIG. 2 depicts an embodiment of a thermal cycler 1 having a gradient block 2 and a touchscreen graphical display and interface 3. The top of the gradient block contains holes 4 for sample tubes containing PCR reaction mixtures. A heated lid 5 surmounts the gradient block and, when in a closed position, prevents the reaction mixture in the tubes from boiling and evaporating. A microprocessor is contained in the thermal cycler for storing data and providing information to the operator.

The thermal cycler of this invention can be used in biochemical and biological reactions other than the PCR reactions described herein. For instance, the thermal cycler may be used for reactions such as the ligase chain reaction, DNA cycle sequencing, DNA synthesis, coupled amplification sequencing, rapid amplification of cDNA ends, and the like. A specific example of the use of the thermal cycler of this invention would be to establish a gradient of 4°C to 20°C for a protein crystallography analysis.

Each of the foregoing patents, patent applications and references that are recited in this application are herein incorporated in their entirety by reference. In accordance with the present invention, it is believed that other modifications, variations and changes can be made by those skilled in the art in view of the teachings set forth herein without departing from the spirit and scope of the invention. It is, therefore, to be understood that all such variations, modifications, and changes are believed to fall within the scope of the present invention as defined by the appended claims. What is claimed is:

Claims

1. A gradient block, comprising: a top plate; and a data storage device, wherein the data storage device is adapted to be connected to a control unit and stores data related to the calibration of the gradient block.

2. The gradient block of claim 1, further comprising one of a heater and a cooler.

3. The gradient block of claim 2, further comprising a heat sink.

4. The gradient block of claim 2, wherein the one of a heater and a cooler comprises a thermoelectric device.

5. The gradient block of claim 1, wherein the storage device comprises non-volatile memory.

6. The gradient block at claim 1, which data related to the calibration of the block includes data about the thermal characteristics of the block.

7. An apparatus for generating a temperature gradient across a gradient block, comprising: a gradient block, comprising a top plate, and at least four pairs of thermoelectric devices in thermal contact with the top plate, said pairs of thermoelectric devices being capable of independently transferring heat with the top plate and dividing the top plate into at least four heat transfer zones; and a control unit for controlling the temperature of the thermoelectric devices, said control unit containing a microprocessor capable of storing data.

8. The apparatus of claim 7, wherein the top plate includes a top surface having holes for holding sample tubes.

9. The apparatus of claim 7, wherein the thermoelectric devices are capable of cooling and heating the gradient block.

10. The apparatus of claim 7, wherein the thermoelectric devices are Peltier devices.

11. The apparatus of claim 7, wherein the gradient block includes temperature sensors.

12. The apparatus of claim 7, wherein the gradient block contains a data storage device which is capable of storing data concerning the thermal characteristics performance of the block.

13. The apparatus of claim 12, wherein the data storage device comprises nonvolatile memory.

14. The apparatus of claim 7, wherein the microprocessor contains data on PCR reactions.

15. The apparatus of claim 7, wherein the electronic control unit comprises a graphical display for displaying the block temperature in real time.

16. The apparatus of claim 15, wherein the graphical display includes a touchscreen which permits the user to input data by touching icons on the computer screen.

17. The apparatus of claim 7, wherein the top plate is fabricated from a metal.

18. The apparatus of claim 17, wherein the metal is selected from the group consisting of aluminum, brass and silver.

19. The apparatus of claim 7, wherein the gradient block is enclosed in a casing including a lid designed to cover sample tubes inserted in the top plate.

20. The apparatus of claim 7, wherein the sample tubes contain a liquid sample of nucleic acid.

21. The apparatus of claim 7, wherein the microprocessor is programmed to perform a PCR reaction.

22. The apparatus of claim 7, wherein the microprocessor is programmed to perform a protein crystallography reaction.

23. A method for replacing a gradient block in a thermal cycler, said thermal cycler comprising a computer and a first gradient block, said method comprising: removing a first gradient block from the thermal cycler; connecting a second gradient block comprising a second data storage device containing thermal calibration data; and modifying a thermal control algorithm of the computer to reflect the thermal characteristics of the second gradient block.

24. The method of claim 23, wherein the data storage device comprises non- volatile memory.

25. The method of claim 23, wherein the second gradient block is calibrated using an external temperature probe.

26. A method of using a thermal cycler comprising: installing a gradient block onto the thermal cycler; providing data stored on a data storage device connected to the gradient block to a control unit connected to the thermal cycler; and controlling at least one heat transfer device connected to the gradient block based on the data provided.